Epigenetics in multiple myeloma: From mechanisms to therapy.

Multiple myeloma (MM) is a tumor of antibody producing plasmablasts/plasma cells that resides within the bone marrow (BM). In addition to the well-established role of genetic lesions and tumor-microenvironment interactions in the development of MM, deregulated epigenetic mechanisms are emerging as important in MM pathogenesis. Recently, MM sequencing and expression projects have revealed that mutations and copy number variations as well as deregulation in the expression of epigenetic modifiers are characteristic features of MM. In the past decade, several studies have suggested epigenetic mechanisms via DNA methylation, histone modifications and non-coding RNAs as important contributing factors in MM with impacts on disease initiation, progression, clonal heterogeneity and response to treatment. Herein we review the present view and knowledge that has accumulated over the past decades on the role of epigenetics in MM, with focus on the interplay between epigenetic mechanisms and the potential use of epigenetic inhibitors as future treatment modalities for MM.

SUMO-modified insulin-like growth factor 1 receptor (IGF-1R) increases cell cycle progression and cell proliferation.

Increasing number of studies have shown nuclear localization of the insulin-like growth factor 1 receptor (nIGF-1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF-1R have, however, still not been disclosed. Previously, we reported that IGF-1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple-SUMO-site-mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R-). Cell clones (R-WT and R-TSM) expressing equal amounts of IGF-1R were selected for experiments. Phosphorylation of IGF-1R, Akt, and Erk upon IGF-1 stimulation was equal in R-WT and R-TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R-WT proliferated substantially faster than R-TSM, which did not differ significantly from the empty vector control. Upon IGF-1 stimulation G1-S-phase progression of R-WT increased from 12 to 38%, compared to 13 to 20% of R-TSM. The G1-S progression of R-WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO-IGF-1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO-IGF-1R dependent mechanisms seem important.

A Role for the Chromatin-Remodeling Factor BAZ1A in Neurodevelopment.

Chromatin-remodeling factors are required for a wide range of cellular and biological processes including development and cognition, mainly by regulating gene expression. As these functions would predict, deregulation of chromatin-remodeling factors causes various disease syndromes, including neurodevelopmental disorders. Recent reports have linked mutations in several genes coding for chromatin-remodeling factors to intellectual disability (ID). Here, we used exome sequencing and identified a nonsynonymous de novo mutation in BAZ1A (NM_182648.2:c.4043T > G, p.Phe1348Cys), encoding the ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1), in a patient with unexplained ID. ACF1 has been previously reported to bind to the promoter of the vitamin D receptor (VDR)-regulated genes and suppress their expression. Our results show that the patient displays decreased binding of ACF1 to the promoter of the VDR-regulated gene CYP24A1. Using RNA sequencing, we find that the mutation affects the expression of genes involved in several pathways including vitamin D metabolism, Wnt signaling and synaptic formation. RNA sequencing of BAZ1A knockdown cells and Baz1a knockout mice revealed that BAZ1A carry out distinctive functions in different tissues. We also demonstrate that BAZ1A depletion influence the expression of genes important for nervous system development and function. Our data point to an important role for BAZ1A in neurodevelopment, and highlight a possible link for BAZ1A to ID.

IGF-1 suppresses Bim expression in multiple myeloma via epigenetic and posttranslational mechanisms.

Insulin-like growth factor-1 (IGF-1) is an important growth and survival factor in multiple myeloma (MM). Here, we demonstrate that IGF-1 induces significant down-regulation of the proapoptotic BH3-only protein Bim in MM cells. Reduced Bim levels by RNA interference (RNAi) protected cells from drug-induced cell death. The IGF-1-mediated down-regulation of Bim was the result of (1) reduced transcription by activation of the Akt pathway and inactivation of the transcription factor FoxO3a, (2) increased proteasome-mediated degradation of the Bim extra-long protein by activation of the mitogen-activated protein kinase pathway, and (3) epigenetic regulation of both the Bim and the FoxO3a promoter. Treatment of cells with the histone deacetylase inhibitor LBH589 resulted in a clear up-regulation in the expression of Bim. Furthermore, the methylation inhibitor 5-aza-2'deoxycytidine (decitabine) significantly increased the effects of LBH589. On IGF-1 treatment, the Bim promoter region was found to be unmethylated, whereas chromatin immunoprecipitation analysis of the IGF-1-treated cells showed both a reduced histone H3 tail Lys9 (H3K9) acetylation and an increased H3K9 dimethylation, which contributed actively to its silencing. These data identify a new mechanism in the IGF-1-dependent survival of MM cells and emphasize the need for IGF-1-targeted drug therapy.

Mantle cell lymphoma displays a homogenous methylation profile: a comparative analysis with chronic lymphocytic leukemia.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are mature CD5(+) B-cell malignancies with different biological/clinical characteristics. We recently reported an association between different prognostic subgroups of CLL (i.e., IGHV mutated and unmutated) and genomic methylation pattern. However, the relationship between DNA methylation and prognostic markers, such as the proliferation gene expression signature, has not been investigated in MCL. We applied high-resolution methylation microarrays (27,578 CpG sites) to assess the global DNA methylation profiles in 20 MCL (10 each with high/low proliferation signature) and 30 CLL (15 poor-prognostic IGHV unmutated subset #1 and 15 good-prognostic IGHV mutated subset #4) samples. Notably, MCL and each CLL subset displayed distinct genomic methylation profiles. After unsupervised hierarchical clustering, 17/20 MCL cases formed a cluster separate from CLL, while CLL subsets #1 and #4 formed subclusters. Surprisingly, few differentially methylated genes (n = 6) were identified between high vs. low proliferation MCL. In contrast, distinct methylation profiles were demonstrated for MCL and CLL. Importantly, certain functional classes of genes were preferentially methylated in either disease. For instance, developmental genes, in particular homeobox transcription factor genes (e.g., HLXB9, HOXA13), were more highly methylated in MCL, whereas apoptosis-related genes were enriched among targets methylated in CLL (e.g., CYFIP2, NR4A1). Results were validated using pyrosequencing, RQ-PCR and reexpression of specific genes. In summary, the methylation profile of MCL was homogeneous and no correlation with the proliferation signature was observed. Compared to CLL, however, marked differences were discovered such as the preferential methylation of homeobox genes in MCL.

Dormant SOX9-Positive Cells Facilitate MYC-Driven Recurrence of Medulloblastoma.

Relapse is the leading cause of death in patients with medulloblastoma, the most common malignant pediatric brain tumor. A better understanding of the mechanisms underlying recurrence could lead to more effective therapies for targeting tumor relapses. Here, we observed that SOX9, a transcription factor and stem cell/glial fate marker, is limited to rare, quiescent cells in high-risk medulloblastoma with MYC amplification. In paired primary-recurrent patient samples, SOX9-positive cells accumulated in medulloblastoma relapses. SOX9 expression anti-correlated with MYC expression in murine and human medulloblastoma cells. However, SOX9-positive cells were plastic and could give rise to a MYC high state. To follow relapse at the single-cell level, an inducible dual Tet model of medulloblastoma was developed, in which MYC expression was redirected in vivo from treatment-sensitive bulk cells to dormant SOX9-positive cells using doxycycline treatment. SOX9 was essential for relapse initiation and depended on suppression of MYC activity to promote therapy resistance, epithelial-mesenchymal transition, and immune escape. p53 and DNA repair pathways were downregulated in recurrent tumors, whereas MGMT was upregulated. Recurrent tumor cells were found to be sensitive to treatment with an MGMT inhibitor and doxorubicin. These findings suggest that recurrence-specific targeting coupled with DNA repair inhibition comprises a potential therapeutic strategy in patients affected by medulloblastoma relapse. SIGNIFICANCE: SOX9 facilitates therapy escape and recurrence in medulloblastoma via temporal inhibition of MYC/MYCN genes, revealing a strategy to specifically target SOX9-positive cells to prevent tumor relapse.

Over-accumulation of nuclear IGF-1 receptor in tumor cells requires elevated expression of the receptor and the SUMO-conjugating enzyme Ubc9.

The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in tumor cell growth and is overexpressed in many cancers. IGF-1R's trans-membrane kinase signaling pathways have been well characterized. Very recently, we showed that SUMOylation mediates nuclear translocation of the IGF-1R, and that nuclear IGF-1R (nIGF-1R) binds to enhancer regions and activates transcription. We identified three lysine residues in the ß-subunit of the receptor and that mutation of these blocks nuclear translocation and gene activation. Furthermore, accumulation of nIGF-1R was proven strongly dependent on the specific SUMO-conjugating enzyme Ubc9. Here we show that nIGF-1R originates solely from the cell membrane and that phosphorylation of the core tyrosine residues of the receptor kinase is crucial for nuclear accumulation. We also compared the levels of nIGF-1R, measured as nuclear/membrane ratios, in tumor and normal cells. We found that the breast cancer cell line MCF-7 has 13-fold higher amounts of nIGF-1R than breast epithelial cells (IME) which showed only a small amount of nIGF-1R. In comparison, the total expression of IGF-1R was only 3.7- higher in MCF-7. Comparison of several other tumor and normal cell lines showed similar tumor cell over-accumulation of nIGF-1R, exceeding the total receptor expression substantially. Ectopic overexpression (>10-fold) of the receptor increased nIGF-1R in IME cells but not to that high level as in wild type MCF-7. The levels of Ubc9 were higher in all tumor cell lines, compared to the normal cells, and this probably contributes to over-accumulation of nIGF-1R. Over-accumulation of nIGF-1R may contribute to deregulated gene expression and therewith play a pathophysiological role in cancer cells.

One Omics Approach Does Not Rule Them All: The Metabolome and the Epigenome Join Forces in Haematological Malignancies.

Aberrant DNA methylation, dysregulation of chromatin-modifying enzymes, and microRNAs (miRNAs) play a crucial role in haematological malignancies. These epimutations, with an impact on chromatin accessibility and transcriptional output, are often associated with genomic instability and the emergence of drug resistance, disease progression, and poor survival. In order to exert their functions, epigenetic enzymes utilize cellular metabolites as co-factors and are highly dependent on their availability. By affecting the expression of metabolic enzymes, epigenetic modifiers may aid the generation of metabolite signatures that could be utilized as targets and biomarkers in cancer. This interdependency remains often neglected and poorly represented in studies, despite well-established methods to study the cellular metabolome. This review critically summarizes the current knowledge in the field to provide an integral picture of the interplay between epigenomic alterations and the cellular metabolome in haematological malignancies. Our recent findings defining a distinct metabolic signature upon response to enhancer of zeste homolog 2 (EZH2) inhibition in multiple myeloma (MM) highlight how a shift of preferred metabolic pathways may potentiate novel treatments. The suggested link between the epigenome and the metabolome in haematopoietic tumours holds promise for the use of metabolic signatures as possible biomarkers of response to treatment.

A distinct metabolic response characterizes sensitivity to EZH2 inhibition in multiple myeloma.

Multiple myeloma (MM) is a heterogeneous haematological disease that remains clinically challenging. Increased activity of the epigenetic silencer EZH2 is a common feature in patients with poor prognosis. Previous findings have demonstrated that metabolic profiles can be sensitive markers for response to treatment in cancer. While EZH2 inhibition (EZH2i) has proven efficient in inducing cell death in a number of human MM cell lines, we hereby identified a subset of cell lines that despite a global loss of H3K27me3, remains viable after EZH2i. By coupling liquid chromatography-mass spectrometry with gene and miRNA expression profiling, we found that sensitivity to EZH2i correlated with distinct metabolic signatures resulting from a dysregulation of genes involved in methionine cycling. Specifically, EZH2i resulted in a miRNA-mediated downregulation of methionine cycling-associated genes in responsive cells. This induced metabolite accumulation and DNA damage, leading to G2 arrest and apoptosis. Altogether, we unveiled that sensitivity to EZH2i in human MM cell lines is associated with a specific metabolic and gene expression profile post-treatment.

Epigenetic silencing of the interferon regulatory factor ICSBP/IRF8 in human multiple myeloma.

OBJECTIVE: Multiple myeloma (MM) is presently an incurable malignant plasma cell tumor. The objective of this study was to investigate expression of the interferon regulatory factor family (IRF1-9) and the potential role of DNA methylation in silencing IRF genes in MM cell lines and purified MM cells from patients. MATERIALS AND METHODS: Using a panel of 13 human MM cell lines and purified CD138+ cells from nine MM patients, expression of IRF genes was investigated by quantitative reverse transcriptase polymerase chain reaction and Western blot. DNA methylation of the interferon consensus sequence-binding protein (ICSBP/IRF8) gene was measured using pyrosequencing, and the effect of promoter methylation on expression was analyzed by in vitro methylation of a cloned ICSBP/IRF8 promoter, and treatment of MM cells with 5-aza-2'-deoxycytidine (DAC). RESULTS: Eight of thirteen of the MM cell lines were found to lack ICSBP/IRF8 expression, associated with hypermethylation of the CpG island in the ICSBP/IRF8 promoter. We also found that ICSBP/IRF8 was significantly underexpressed in primary MM cells, whereas the ICSBP/IRF8 promoter was methylated in only one of nine of primary purified CD138+ MM samples. DAC-mediated demethylation restored endogenous ICSBP/IRF8 expression, whereas in vitro methylation silenced the promoter. CONCLUSION: Expression of the ICSBP/IRF8 gene is silenced in a majority of MM cell lines and primary CD138+ MM cells. DNA methylation of the ICSBP/IRF8 gene is a frequent event in MM cell lines, but silencing is also observed in the absence of methylation. These results suggest that silencing of ICSBP/IRF8 expression, by DNA methylation or other epigenetic mechanisms, may be associated with the malignant phenotype of MM.

Expression of erythropoietin receptor and in vitro functional effects of epoetins in B-cell malignancies.

PURPOSE: Erythropoietin (EPO) and EPO receptor (EPO-R) expression have been reported in solid tumors and are claimed to regulate tumor growth; however, no data have been published on this issue in B-cell malignancies or normal lymphoid cells. This report describes genomic/protein EPO-R expression and in vitro effects of recombinant human EPO (epoetin) in B-cell chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL), and multiple myeloma (MM). EXPERIMENTAL DESIGN: Blood samples were obtained from patients with B-CLL, MCL, and healthy volunteers, and bone marrow was obtained from MM patients. EPO-R mRNA was detected by reverse transcription-PCR. EPO-R surface expression was investigated by flow cytometry using digoxigenin-labeled epoetin and polyclonal rabbit anti-EPO-R antibody for intracellular receptor. Tumor cell stimulation was determined in vitro using [(3)H]thymidine incorporation and CD69 expression after exposure to epoetin alpha or beta or darbepoetin alpha. RESULTS: EPO-R mRNA was detected in mononuclear cells from 32 of 41 (78%) B-CLL and 5 of 7 (71%) MCL patients, and 21 of 21 (100%) MM samples. Expression was also detected in highly purified T cells from six of eight B-CLL patients, four of four MM patients, and normal donor B and T cells. Surface EPO-R protein was not detected. Intracellular EPO-R staining with anti-EPO-R antibodies was unspecific. No tumor-stimulatory effect was observed with high epoetin concentrations. CONCLUSIONS: EPO-R gene is frequently expressed in lymphoid malignancies and normal B and T cells. However, there was no surface protein expression and no epoetin-induced in vitro stimulation of tumor B cells, indicating that epoetin therapy in vivo is likely to be safe in patients with lymphoid malignancies.

Targeting EZH2 in Multiple Myeloma-Multifaceted Anti-Tumor Activity.

The enhancer of zeste homolog 2 (EZH2) is the enzymatic subunit of the polycomb repressive complex 2 (PRC2) that exerts important functions during normal development as well as disease. PRC2 through EZH2 tri-methylates histone H3 lysine tail residue 27 (H3K27me3), a modification associated with repression of gene expression programs related to stem cell self-renewal, cell cycle, cell differentiation, and cellular transformation. EZH2 is deregulated and subjected to gain of function or loss of function mutations, and hence functions as an oncogene or tumor suppressor gene in a context-dependent manner. The development of highly selective inhibitors against the histone methyltransferase activity of EZH2 has also contributed to insight into the role of EZH2 and PRC2 in tumorigenesis, and their potential as therapeutic targets in cancer. EZH2 can function as an oncogene in multiple myeloma (MM) by repressing tumor suppressor genes that control apoptosis, cell cycle control and adhesion properties. Taken together these findings have raised the possibility that EZH2 inhibitors could be a useful therapeutic modality in MM alone or in combination with other targeted agents in MM. Therefore, we review the current knowledge on the regulation of EZH2 and its biological impact in MM, the anti-myeloma activity of EZH2 inhibitors and their potential as a targeted therapy in MM.

The miR-125a and miR-320c are potential tumor suppressor microRNAs epigenetically silenced by the polycomb repressive complex 2 in multiple myeloma.

We have previously presented the histone methyltransferase enhancer of zeste homolog 2 (EZH2) of the polycomb repressive complex 2 (PRC2) as a potential therapeutic target in Multiple Myeloma (MM). In a recent article in Oncotarget by Alzrigat. et al. 2017, we have reported on the novel finding that EZH2 inhibition using the highly selective inhibitor of EZH2 enzymatic activity, UNC1999, reactivated the expression of microRNA genes previously reported to be underexpressed in MM. Among these, we have identified miR-125a-3p and miR-320c as potential tumor suppressor microRNAs as they were predicted to target MM-associated oncogenes; IRF-4, XBP-1 and BLIMP-1. We also found EZH2 inhibition to reactivate the expression of miR-494, a previously reported regulator of the c-MYC oncogene. In addition, we could report that EZH2 inhibition downregulated the expression of a few well described oncogenic microRNAs in MM. The data from our recent article are here highlighted as it shed a new light onto the oncogenic function of the PRC2 in MM. These data further strengthen the notion that the PRC2 complex may be of potential therapeutic interest.

The polycomb group protein BMI-1 inhibitor PTC-209 is a potent anti-myeloma agent alone or in combination with epigenetic inhibitors targeting EZH2 and the BET bromodomains.

Multiple myeloma (MM) is a tumor of plasmablasts/plasma cells (PCs) characterized by the expansion of malignant PCs with complex genetic aberrations in the bone marrow (BM). Recent reports, by us and others, have highlighted the polycomb group (PcG) proteins as potential targets for therapy in MM. The PcG protein BMI-1 of the polycomb repressive complex 1 (PRC1) has been reported to be overexpressed and to possess oncogenic functions in MM. Herein, we report on the anti-myeloma effects of the BMI-1 inhibitor PTC-209 and demonstrate that PTC-209 is a potent anti-myeloma agent in vitro using MM cell lines and primary MM cells. We show that PTC-209 reduces the viability of MM cells via induction of apoptosis and reveal that the anti-MM actions of PTC-209 are mediated by on-target effects i.e. downregulation of BMI-1 protein and the associated repressive histone mark H2AK119ub, leaving other PRC1 subunits such as CBX-7 and the catalytic subunit RING1B unaffected. Importantly, we demonstrate that PTC-209 exhibits synergistic and additive anti-myeloma activity when combined with other epigenetic inhibitors targeting EZH2 and BET bromodomains. Collectively, these data qualify BMI-1 as a candidate for targeted therapy in MM alone or in combinations with epigenetic inhibitors directed to PRC2/EZH2 or BET bromodomains.

EZH2 inhibition in multiple myeloma downregulates myeloma associated oncogenes and upregulates microRNAs with potential tumor suppressor functions.

Multiple Myeloma (MM) is a plasma cell tumor localized to the bone marrow (BM). Despite the fact that current treatment strategies have improved patients' median survival time, MM remains incurable. Epigenetic aberrations are emerging as important players in tumorigenesis making them attractive targets for therapy in cancer including MM. Recently, we suggested the polycomb repressive complex 2 (PRC2) as a common denominator of gene silencing in MM and presented the PRC2 enzymatic subunit enhancer of zeste homolog 2 (EZH2) as a potential therapeutic target in MM. Here we further dissect the anti-myeloma mechanisms mediated by EZH2 inhibition and show that pharmacological inhibition of EZH2 reduces the expression of MM-associated oncogenes; IRF-4, XBP-1, PRDM1/BLIMP-1 and c-MYC. We show that EZH2 inhibition reactivates the expression of microRNAs with tumor suppressor functions predicted to target MM-associated oncogenes; primarily miR-125a-3p and miR-320c. ChIP analysis reveals that miR-125a-3p and miR-320c are targets of EZH2 and H3K27me3 in MM cell lines and primary cells. Our results further highlight that polycomb-mediated silencing in MM includes microRNAs with tumor suppressor activity. This novel role strengthens the oncogenic features of EZH2 and its potential as a therapeutic target in MM.

Genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in multiple myeloma reveals the importance of Polycomb gene targeting and highlights EZH2 as a potential therapeutic target.

Multiple myeloma (MM) is a malignancy of the antibody-producing plasma cells. MM is a highly heterogeneous disease, which has hampered the identification of a common underlying mechanism for disease establishment as well as the development of targeted therapy. Here we present the first genome-wide profiling of histone H3 lysine 27 and lysine 4 trimethylation in MM patient samples, defining a common set of active H3K4me3-enriched genes and silent genes marked by H3K27me3 (H3K27me3 alone or bivalent) unique to primary MM cells, when compared to normal bone marrow plasma cells. Using this epigenome profile, we found increased silencing of H3K27me3 targets in MM patients at advanced stages of the disease, and the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also demonstrated that pharmacological inhibition of EZH2 had anti-myeloma effects in both MM cell lines and CD138+ MM patient cells. In addition, EZH2 inhibition decreased the global H3K27 methylation and induced apoptosis. Taken together, these data suggest an important role for the Polycomb repressive complex 2 (PRC2) in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM.

Growth signals employ CGGBP1 to suppress transcription of Alu-SINEs.

CGGBP1 (CGG triplet repeat-binding protein 1) regulates cell proliferation, stress response, cytokinesis, telomeric integrity and transcription. It could affect these processes by modulating target gene expression under different conditions. Identification of CGGBP1-target genes and their regulation could reveal how a transcription regulator affects such diverse cellular processes. Here we describe the mechanisms of differential gene expression regulation by CGGBP1 in quiescent or growing cells. By studying global gene expression patterns and genome-wide DNA-binding patterns of CGGBP1, we show that a possible mechanism through which it affects the expression of RNA Pol II-transcribed genes in trans depends on Alu RNA. We also show that it regulates Alu transcription in cis by binding to Alu promoter. Our results also indicate that potential phosphorylation of CGGBP1 upon growth stimulation facilitates its nuclear retention, Alu-binding and dislodging of RNA Pol III therefrom. These findings provide insights into how Alu transcription is regulated in response to growth signals.

Increased resistance to proteasome inhibitors in multiple myeloma mediated by cIAP2--implications for a combinatorial treatment.

Despite the introduction of new treatment options for multiple myeloma (MM), a majority of patients relapse due to the development of resistance. Unraveling new mechanisms underlying resistance could lead to identification of possible targets for combinatorial treatment. Using TRAF3 deleted/mutated MM cell lines, we evaluated the role of the cellular inhibitor of apoptosis 2 (cIAP2) in drug resistance and uncovered the plausible mechanisms underlying this resistance and possible strategies to overcome this by combinatorial treatment. In MM, cIAP2 is part of the gene signature of aberrant NF-κB signaling and is heterogeneously expressed amongst MM patients. In cIAP2 overexpressing cells a decreased sensitivity to the proteasome inhibitors bortezomib, MG132 and carfilzomib was observed. Gene expression analysis revealed that 440 genes were differentially expressed due to cIAP2 overexpression. Importantly, the data imply that cIAPs are rational targets for combinatorial treatment in the population of MM with deleted/mutated TRAF3. Indeed, we found that treatment with the IAP inhibitor AT-406 enhanced the anti-MM effect of bortezomib in the investigated cell lines. Taken together, our results show that cIAP2 is an important factor mediating bortezomib resistance in MM cells harboring TRAF3 deletion/mutation and therefore should be considered as a target for combinatorial treatment.

Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia.

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

The IGF-1 receptor inhibitor picropodophyllin potentiates the anti-myeloma activity of a BH3-mimetic.

The ABT-analogous 737, 263 and 199 are BH3 mimetics showing potent anti-myeloma (MM) activity, but only on defined molecular subgroups of MM patients presenting a Bcl-2high/Mcl-1low profile. IGF-1 is a major survival factor in MM regulating the expression of Bcl-2 proteins and might therefore be a resistance factor to these ABT-analogous. We first show that IGF-1 protected human MM cell lines (HMCLs) against ABT-737. Concurrently, the IGF-1 receptor inhibitor picropodophyllin (PPP) synergistically sensitized HMCL, primary human MM and murine 5T33MM cells to ABT-737 and ABT-199 by further decreasing cell viability and enhancing apoptosis. Knockdown of Bcl-2 by shRNA protected MM cells to ABT-737, while Mcl-1 shRNA sensitized the cells. PPP overcame the Bcl-2 dependency of ABT-737, but failed to completely overcome the protective effect of Mcl-1. In vivo, co-treatment of 5T33MM bearing mice significantly decreased tumor burden and prolonged overall survival both in a prophylactic and therapeutic setting. Interestingly, proteasome inhibitor resistant CD138- 5T33MM cells were more sensitive to ABT-737, whereas PPP alone targeted the CD138+ cells more effectively. After co-treatment, both subpopulations were targeted equally. Together, the combination of an IGF-1R inhibitor and an ABT-analogue displays synergistic anti-myeloma activity providing the rational for further (pre)clinical testing.